Discovery of Biologically Active Oncologic and Immunologic Small Molecule Therapies using Zebrafish: Overview and Example of Modulation of T Cell Activation

Nikolaus S. Trede1, William Heaton2, Suzanne Ridges2, Hossein Sofla2, Matthew Cusick3, David Bearss2, David Jones2, Robert S. Fujinami3

1 Department of Pediatrics, University of Utah, Salt Lake City, Utah, 2 Department of Oncological Sciences, Huntsman Cancer Institute, Salt Lake City, Utah, 3 Department of Pathology, University of Utah, Salt Lake City, Utah
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.24
DOI:  10.1002/0471141755.ph1424s60
Online Posting Date:  March, 2013
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Abstract

Zebrafish models continue to gain popularity as in vivo models for drug discovery. Described in this overview are advantages and challenges of zebrafish drug screening, as well as a novel in vivo screen for immunomodulatory compounds using transgenic, T cell reporting zebrafish larvae designed for discovery of compounds targeting T cell leukemia. This assay system allows rapid screening of large numbers of compounds while avoiding the pitfalls of assays based on cell cultures, which lack biologic context and are afflicted by genomic instability. The rationale for this approach is based on similarities of immature normal T cells and developmentally arrested, malignant lymphoblasts in mammalian species. The screening algorithm has been used to identify a nontoxic compound with activity in both acute leukemia models and models of multiple sclerosis, demonstrating the utility of this screening procedure. Curr. Protoc. Pharmacol. 60:14.24.1‐14.24.13. © 2013 by John Wiley & Sons, Inc.

Keywords: zebrafish; leukemia; immunomodulatory agents; multiple sclerosis; melanoma; pancreatic carcinoma; liver cancer; rhabdomyosarcoma; testicular cancer; small molecule drug screening

     
 
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Table of Contents

  • Challenges in Drug Discovery Using In Vitro Cell‐Based Screening
  • Zebrafish as a Model for Human Disease and Drug Screening
  • Need for Targeted Therapies to Treat Immune‐Mediated Oncologic Diseases—Use of Zebrafish to Identify Immunomodulatory Agents
  • A Zebrafish Screen for Immunomodulatory Compounds
  • Summary
  • Acknowledgements
  • Literature Cited
  • Figures
  • Tables
     
 
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Literature Cited

Literature Cited
   Adams, K.N., Takaki, K., Connolly, L.E., Wiedenhoft, H., Winglee, K., Humbert, O., Edelstein, P.H., Cosma, C.L., and Ramakrishnan, L. 2011. Drug tolerance in replicating mycobacteria mediated by a macrophage‐induced efflux mechanism. Cell 145:39‐53.
   Ali, S., Champagne, D.L., Spaink, H.P., and Richardson, M.K. 2011. Zebrafish embryos and larvae: A new generation of disease models and drug screens. Birth Defects Res. C Embryo Today 93:115‐133.
   Amsterdam, A., Lai, K., Komisarczuk, A.Z., Becker, T.S., Bronson, R.T., Hopkins, N., and Lees, J.A. 2009. Zebrafish Hagoromo mutants up‐regulate fgf8 postembryonically and develop neuroblastoma. Mol. Cancer Res. 7:841‐850.
   Anelli, V., Santoriello, C., Distel, M., Koster, R.W., Ciccarelli, F.D., and Mione, M. 2009. Global repression of cancer gene expression in a zebrafish model of melanoma is linked to epigenetic regulation. Zebrafish 6:417‐424.
   Bajoghli, B., Aghaallaei, N., Hess, I., Rode, I., Netuschil, N., Tay, B.H., Venkatesh, B., Yu, J.K., Kaltenbach, S.L., Holland, N.D., Diekhoff, D., Happe, C., Schorpp, M., and Boehm, T. 2009. Evolution of genetic networks underlying the emergence of thymopoiesis in vertebrates. Cell 138:186‐197.
   Boehm, T., Scheu, S., Pfeffer, K., and Bleul, C.C. 2003. Thymic medullary epithelial cell differentiation, thymocyte emigration, and the control of autoimmunity require lympho‐epithelial cross talk via LTbetaR. J. Exp. Med. 198:757‐769.
   Bostrom, B.C., Sensel, M.R., Sather, H.N., Gaynon, P.S., La, M.K., Johnston, K., Erdmann, G.R., Gold, S., Heerema, N.A., Hutchinson, R.J., Provisor, A.J., and Trigg, M.E. 2003. Dexamethasone versus prednisone and daily oral versus weekly intravenous mercaptopurine for patients with standard‐risk acute lymphoblastic leukemia: A report from the Children's Cancer Group. Blood 101:3809‐3817.
   Burdine, L. and Kodadek, T. 2004. Target identification in chemical genetics: The (often) missing link. Chem. Biol. 11:593‐597.
   Burns, C.E., Galloway, J.L., Smith, A.C., Keefe, M.D., Cashman, T.J., Paik, E.J., Mayhall, E.A., Amsterdam, A.H., and Zon, L.I. 2009. A genetic screen in zebrafish defines a hierarchical network of pathways required for hematopoietic stem cell emergence. Blood 113:5776‐5782.
   Ceol, C.J., Houvras, Y., Jane‐Valbuena, J., Bilodeau, S., Orlando, D.A., Battisti, V., Fritsch, L., Lin, W.M., Hollmann, T.J., Ferre, F., Bourque, C., Burke, C.J., Turner, L., Uong, A., Johnson, L.A., Beroukhim, R., Mermel, C.H., Loda, M., Ait‐Si‐Ali, S., Garraway, L.A., Young, R.A., and Zon, L.I. 2011. The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset. Nature 471:513‐517.
   Chen, J., Jette, C., Kanki, J.P., Aster, J.C., Look, A.T., and Griffin, J.D. 2007. NOTCH1‐induced T‐cell leukemia in transgenic zebrafish. Leukemia 21:462‐471.
   Creton, R. 2004. The calcium pump of the endoplasmic reticulum plays a role in midline signaling during early zebrafish development. Brain Res. Dev. Brain Res. 151:33‐41.
   Cusick, M.F., Libbey, J.E., Trede, N.S., Eckels, D.D., and Fujinami, R.S. 2012. Human T cell expansion and experimental autoimmune encephalomyelitis inhibited by Lenaldekar, a small molecule discovered in a zebrafish screen. J. Neuroimmunol. 244:35‐44.
   Davidson, A.J. and Zon, L.I. 2004. The ‘definitive’ (and ‘primitive’) guide to zebrafish hematopoiesis. Oncogene 23:7233‐7246.
   Druker, B.J., Talpaz, M., Resta, D.J., Peng, B., Buchdunger, E., Ford, J.M., Lydon, N.B., Kantarjian, H., Capdeville, R., Ohno‐Jones, S., and Sawyers, C.L. 2001. Efficacy and safety of a specific inhibitor of the BCR‐ABL tyrosine kinase in chronic myeloid leukemia. N. Engl. J. Med. 344:1031‐1037.
   Feng, H., Langenau, D.M., Madge, J.A., Quinkertz, A., Gutierrez, A., Neuberg, D.S., Kanki, J.P., and Look, A.T. 2007. Heat‐shock induction of T‐cell lymphoma/leukaemia in conditional Cre/lox‐regulated transgenic zebrafish. Br. J. Haematol. 138:169‐175.
   Feng, Y., Santoriello, C., Mione, M., Hurlstone, A., and Martin, P. 2010. Live imaging of innate immune cell sensing of transformed cells in zebrafish larvae: Parallels between tumor initiation and wound inflammation.PLoS Biol. 8:e100562.
   Ferrando, A.A. and Look, A.T. 2000. Clinical implications of recurring chromosomal and associated molecular abnormalities in acute lymphoblastic leukemia. Semin. Hematol. 37:381‐395.
   Frazer, J.K., Meeker, N.D., Rudner, L., Bradley, D.F., Smith, A.C., Demarest, B., Joshi, D., Locke, E.E., Hutchinson, S.A., Tripp, S., Perkins, S.L., and Trede, N.S. 2009. Heritable T‐cell malignancy models established in a zebrafish phenotypic screen. Leukemia 23:1825‐1835.
   Gutierrez, A., Grebliunaite, R., Feng, H., Kozakewich, E., Zhu, S., Guo, F., Payne, E., Mansour, M., Dahlberg, S.E., Neuberg, D.S., den Hertog, J., Prochownik, E.V., Testa, J.R., Harris, M., Kanki, J.P., and Look, A.T. 2011. Pten mediates Myc oncogene dependence in a conditional zebrafish model of T cell acute lymphoblastic leukemia. J. Exp. Med. 208:1595‐1603.
   Haggarty, S.J., Mayer, T.U., Miyamoto, D.T., Fathi, R., King, R.W., Mitchison, T.J., and Schreiber, S.L. 2000. Dissecting cellular processes using small molecules: Identification of colchicine‐like, taxol‐like and other small molecules that perturb mitosis. Chem. Biol. 7:275‐286.
   Haxhinasto, S., Mathis, D., and Benoist, C. 2008. The AKT‐mTOR axis regulates de novo differentiation of CD4+Foxp3+ cells. J. Exp. Med. 205:565‐574.
   Kamal, A., Reddy, K.S., Khan, M.N., Shetti, R.V., Ramaiah, M.J., Pushpavalli, S.N., Srinivas, C., Pal‐Bhadra, M., Chourasia, M., Sastry, G.N., Juvekar, A., Zingde, S., and Barkume, M. 2010. Synthesis, DNA‐binding ability and anticancer activity of benzothiazole/benzoxazole‐pyrrolo[2,1‐c][1,4]benzodiazepine conjugates. Bioorg. Med. Chem. 18:4747‐4761.
   Kawakami, K., Abe, G., Asada, T., Asakawa, K., Fukuda, R., Ito, A., Lal, P., Mouri, N., Muto, A., Suster, M.L., Takakubo, H., Urasaki, A., Wada, H., and Yoshida, M. 2010. zTrap: Zebrafish gene trap and enhancer trap database. BMC Dev. Biol. 10:105.
   Kokel, D., Bryan, J., Laggner, C., White, R., Cheung, C.Y., Mateus, R., Healey, D., Kim, S., Werdich, A.A., Haggarty, S.J., Macrae, C.A., Shoichet, B., and Peterson, R.T. 2010. Rapid behavior‐based identification of neuroactive small molecules in the zebrafish. Nat. Chem. Biol. 6:231‐237.
   Lam, S.H., Wu, Y.L., Vega, V.B., Miller, L.D., Spitsbergen, J., Tong, Y., Zhan, H., Govindarajan, K.R., Lee, S., Mathavan, S., Murthy, K.R., Buhler, D.R., Liu, E.T., and Gong, Z. 2006. Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression. Nat. Biotechnol. 24:73‐75.
   Langenau, D.M., Traver, D., Ferrando, A.A., Kutok, J.L., Aster, J.C., Kanki, J.P., Lin, S., Prochownik, E., Trede, N.S., Zon, L.I., and Look, A.T. 2003. Myc‐induced T cell leukemia in transgenic zebrafish. Science 299:887‐890.
   Langenau, D.M., Ferrando, A.A., Traver, D., Kutok, J.L., Hezel, J.P., Kanki, J.P., Zon, L.I., Look, A.T., and Trede, N.S. 2004. In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrafish. Proc. Natl. Acad. Sci. U.S.A. 101:7369‐7374.
   Langenau, D.M., Keefe, M.D., Storer, N.Y., Guyon, J.R., Kutok, J.L., Le, X., Goessling, W., Neuberg, D.S., Kunkel, L.M., and Zon, L.I. 2007. Effects of RAS on the genesis of embryonal rhabdomyosarcoma. Genes Dev. 21:1382‐1395.
   Long, Q., Meng, A., Wang, H., Jessen, J.R., Farrell, M.J., and Lin, S. 1997. GATA‐1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene. Development 124:4105‐4111.
   Medyouf, H., Gao, X., Armstrong, F., Gusscott, S., Liu, Q., Gedman, A.L., Matherly, L.H., Schultz, K.R., Pflumio, F., You, M.J., and Weng, A.P. 2010. Acute T‐cell leukemias remain dependent on Notch signaling despite PTEN and INK4A/ARF loss. Blood 115:1175‐1184.
   Meeker, N.D. and Trede, N.S. 2008. Immunology and zebrafish: Spawning new models of human disease. Dev. Comp. Immunol. 32:745‐757.
   Mione, M.C. and Trede, N.S. 2010. The zebrafish as a model for cancer. Dis. Model Mech. 3:517‐523.
   Murphey, R.D., Stern, H.M., Straub, C.T., and Zon, L.I. 2006. A chemical genetic screen for cell cycle inhibitors in zebrafish embryos. Chem. Biol. Drug Des. 68:213‐219.
   Neumann, J.C., Dovey, J.S., Chandler, G.L., Carbajal, L., and Amatruda, J.F. 2009. Identification of a heritable model of testicular germ cell tumor in the zebrafish. Zebrafish 6:319‐327.
   North, T.E., Goessling, W., Walkley, C.R., Lengerke, C., Kopani, K.R., Lord, A.M., Weber, G.J., Bowman, T.V., Jang, I.H., Grosser, T., Fitzgerald, G.A., Daley, G.Q., Orkin, S.H., and Zon, L.I. 2007. Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis. Nature 447:1007‐1011.
   Onnebo, S.M., Condron, M.M., McPhee, D.O., Lieschke, G.J., and Ward, A.C. 2005. Hematopoietic perturbation in zebrafish expressing a tel‐jak2a fusion. Exp. Hematol. 33:182‐188.
   Palomero, T., Dominguez, M., and Ferrando, A.A. 2008. The role of the PTEN/AKT pathway in NOTCH1‐induced leukemia. Cell Cycle 7:965‐970.
   Pardo‐Martin, C., Chang, T.Y., Koo, B.K., Gilleland, C.L., Wasserman, S.C., and Yanik, M.F. 2010. High‐throughput in vivo vertebrate screening. Nat. Methods 7:634‐636.
   Park, S.W., Davison, J.M., Rhee, J., Hruban, R.H., Maitra, A., and Leach, S.D. 2008. Oncogenic KRAS induces progenitor cell expansion and malignant transformation in zebrafish exocrine pancreas. Gastroenterology 134:2080‐2090.
   Patton, E.E., Widlund, H.R., Kutok, J.L., Kopani, K.R., Amatruda, J.F., Murphey, R.D., Berghmans, S., Mayhall, E.A., Traver, D., Fletcher, C.D., Aster, J.C., Granter, S.R., Look, A.T., Lee, C., Fisher, D.E., and Zon, L.I. 2005. BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma. Curr. Biol. 15:249‐254.
   Peterson, R.T., Link, B.A., Dowling, J.E., and Schreiber, S.L. 2000. Small molecule developmental screens reveal the logic and timing of vertebrate development. Proc. Natl. Acad. Sci. U.S.A. 97:12965‐12969.
   Postlethwait, J.H. 2006. The zebrafish genome: A review and msx gene case study. Genome Dyn. 2:183‐197.
   Pui, C.H., Relling, M.V., and Downing, J.R. 2004. Acute lymphoblastic leukemia. N. Engl. J. Med. 350:1535‐1548.
   Redfern, W.S., Waldron, G., Winter, M.J., Butler, P., Holbrook, M., Wallis, R., and Valentin, J.P. 2008. Zebrafish assays as early safety pharmacology screens: Paradigm shift or red herring? J. Pharmacol. Toxicol. Methods 58:110‐117.
   Renshaw, S.A. and Trede, N.S. 2012. A model 450 million years in the making: Zebrafish and vertebrate immunity. Dis. Model Mech. 5:38‐47.
   Ridges, S., Heaton, W.L., Joshi, D., Choi, H., Eiring, A., Batchelor, L., Choudhry, P., Manos, E.J., Sofla, H., Sanati, A., Welborn, S., Agarwal, A., Spangrude, G.J., Miles, R.R., Cox, J.E., Frazer, J.K., Deininger, M., Balan, K., Sigman, M., Muschen, M., Perova, T., Johnson, R., Montpellier, B., Guidos, C.J., Jones, D.A., and Trede, N.S. 2012. Zebrafish screen identifies novel compound with selective toxicity against leukemia. Blood 119:5621‐5631.
   Rihel, J., Prober, D.A., Arvanites, A., Lam, K., Zimmerman, S., Jang, S., Haggarty, S.J., Kokel, D., Rubin, L.L., Peterson, R.T., and Schier, A.F. 2010. Zebrafish behavioral profiling links drugs to biological targets and rest/wake regulation. Science 327:348‐351.
   Sabaawy, H.E., Azuma, M., Embree, L.J., Tsai, H.J., Starost, M.F., and Hickstein, D.D. 2006. TEL‐AML1 transgenic zebrafish model of precursor B cell acute lymphoblastic leukemia. Proc. Natl. Acad. Sci. U.S.A. 103:15166‐15171.
   Shepard, J.L., Amatruda, J.F., Stern, H.M., Subramanian, A., Finkelstein, D., Ziai, J., Finley, K.R., Pfaff, K.L., Hersey, C., Zhou, Y., Barut, B., Freedman, M., Lee, C., Spitsbergen, J., Neuberg, D., Weber, G., Golub, T.R., Glickman, J.N., Kutok, J.L., Aster, J.C., and Zon, L.I. 2005. A zebrafish bmyb mutation causes genome instability and increased cancer susceptibility. Proc. Natl. Acad. Sci. U.S.A. 102:13194‐13199.
   Stern, H.M., Murphey, R.D., Shepard, J.L., Amatruda, J.F., Straub, C.T., Pfaff, K.L., Weber, G., Tallarico, J.A., King, R.W., and Zon, L.I. 2005. Small molecules that delay S phase suppress a zebrafish bmyb mutant. Nat. Chem. Biol. 1:366‐370.
   Stewart, R.A., Lee, J.S., Lachnit, M., Look, A.T., Kanki, J.P., and Henion, P.D. 2010. Studying peripheral sympathetic nervous system development and neuroblastoma in zebrafish. Methods Cell Biol. 100:127‐152.
   Tobin, D.M., Vary, J.C. Jr., Ray, J.P., Walsh, G.S., Dunstan, S.J., Bang, N.D., Hagge, D.A., Khadge, S., King, M.C., Hawn, T.R., Moens, C.B., and Ramakrishnan, L. 2010. The lta4h locus modulates susceptibility to mycobacterial infection in zebrafish and humans. Cell 140:717‐730.
   Weng, A.P., Ferrando, A.A., Lee, W., Morris, J.P.t., Silverman, L.B., Sanchez‐Irizarry, C., Blacklow, S.C., Look, A.T., and Aster, J.C. 2004. Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 306:269‐271.
   White, R.M., Cech, J., Ratanasirintrawoot, S., Lin, C.Y., Rahl, P.B., Burke, C.J., Langdon, E., Tomlinson, M.L., Mosher, J., Kaufman, C., Chen, F., Long, H.K., Kramer, M., Datta, S., Neuberg, D., Granter, S., Young, R.A., Morrison, S., Wheeler, G.N., and Zon, L.I. 2011. DHODH modulates transcriptional elongation in the neural crest and melanoma. Nature 471:518‐522.
   Yang, H.W., Kutok, J.L., Lee, N.H., Piao, H.Y., Fletcher, C.D., Kanki, J.P., and Look, A.T. 2004. Targeted expression of human MYCN selectively causes pancreatic neuroendocrine tumors in transgenic zebrafish. Cancer Res. 64:7256‐7262.
   Yeh, J.R., Munson, K.M., Chao, Y.L., Peterson, Q.P., Macrae, C.A., and Peterson, R.T. 2008. AML1‐ETO reprograms hematopoietic cell fate by downregulating scl expression. Development 135:401‐410.
   Yeh, J.R., Munson, K.M., Elagib, K.E., Goldfarb, A.N., Sweetser, D.A., and Peterson, R.T. 2009. Discovering chemical modifiers of oncogene‐regulated hematopoietic differentiation. Nat. Chem. Biol. 5:236‐243.
   Zhu, S., Lee, J.S., Guo, F., Shin, J., Perez‐Atayde, A.R., Kutok, J.L., Rodig, S.J., Neuberg, D.S., Helman, D., Feng, H., Stewart, R.A., Wang, W., George, R.E., Kanki, J.P., and Look, A.T. 2012. Activated ALK collaborates with MYCN in neuroblastoma pathogenesis. Cancer Cell 21:362‐373.
   Zhuravleva, J., Paggetti, J., Martin, L., Hammann, A., Solary, E., Bastie, J.N., and Delva, L. 2008. MOZ/TIF2‐induced acute myeloid leukaemia in transgenic fish. Br. J. Haematol. 143:378‐382.
   Zon, L.I. and Peterson, R.T. 2005. In vivo drug discovery in the zebrafish. Nat. Rev. Drug Discov. 4:35‐44.
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